Hydrolysis of scleroproteins with alcoholic hydroxide solution



April 6, 1965 Filed June 20, 1965 E. J. HAWRYLEWICZ ETAL 3,177,197

HYDROLYSIS OF SCLEROPROTEINS WITH ALCOHOLIC HYDROXIDE SOLUTION 4 Sheets-Sheet 1 CURVE l-HOG HAIR m z u NOOH AND ISOPROPYL ALCOHOL (15:25)

CURVE Z-HOG HAIR IN 2 N NOOH AND WATER (751 25) TIME OF HYDROLYSIS, HOU RS RATE OF SOLUBILIZATION OF HOG HAIR (SPEROENT SUSPENSION) FIG.I.

INVENTORS Ervin J. Hcwrylewicz 8 Irene D. Bliudzius ATTORNEYS April 6, 1965 Filed June 20, 1963 J. HAWRYLEWICZ ET AL HYDROXIDE SOLUTION PHENYLALANINE LEUCINE ISOLEUCINE METHIONINE GLUTAMIC ACID THREONINE msnoms 25x25 LYSINE cYSTINE ARGININE WINE surcma E. HYDROLYSIS OF SCLEROPROTEINS WITH ALCOHOLIC AMINO ACID ANALYSIS OF TREATED H06 HAIR 4 Sheets-Sheet 2 FIG.2.

INVENTORS Ervin J. Howrylewicz 8 Irene D. Bliudzius ATTORNEYS A ril 6, 1965 E. J. HAWRYLEWICZ ETAL 3, 7, 9

HYDROLYSIS OF SCLEROPROTEINS WITH ALCOHOLIC HYDROXIDE SOLUTION Filed June 20, 1963 4 Sheets-Sheet 3 muhawti O0 mus-20m mwhazi ON nub-:22 O- INVENTORS Ervin J. Huwrylewicz 8 uzEmm 6.5564

Irene D. Blviudzius AVER AGE WT. g

Aprll 1965 E. J. HAWRYLEWICZ ETAL 3,177,197

HYDROLYSIS OF SCLERQPROTEINS WITH ALCOHOLIC HYDROXIDE SOLUTION Filed June 20, 1963 4 Sheets-Sheet 4 50 l l I WEEKS ON DIET GROWTH RATE OF RATS MAINTAINED ON SOYBEAN MEAL AND MODIFIED HOG HAIR A= SOYBEAN MEAL DIET (l0 PERCENT) B'HOG HAIR DIET (8 PERCENT SBMP AND 2 PERCENT MODIFIED HOG HAIR) FIG.4.

INVENTORS Ervin J. Howrylewicz 8\ Irene D. Bliudzius ATTORNEYS United States Patent 3,177,197 HYDRQLYSIS 0F SCLEROPROTEINS WITH. ALCOHOLEC HYDROXIDE SOLUTION Ervin J. Hawrylewicz and Irene D. Bliudzius, Chicago,

111., assignors, by mesne assignments, to Reliable Packing Company, Chicago, Ill.

Filed June 20, *1963, Ser. No. 293,223 Claims. (Cl. 260-1235) This invention relates to proteins, and more inpar-- ticular to the treatment of keratin.

Various methods are already known for the treatment of keratin, the keratin compound, such as contained in wool, horns, hoofs, feathers, hair and the like. All of these methods depend on hydrolysis, some with acids and some without acids.

The present invention belongs to the latter group and relates to the alkaline hydrolysis of hair. Certain materials are much easier to'hydrolze' than others. For example, hoofs and horns have been treated with acid materials to obtain a hydrolytic product, using such materials as acid or acid chloride, all operating in strictly an acid. medium. Certain other products, such as feathers, have been hydrolyzed by the use of steam-under elevated temperature and pressure toproduce a hydrolyzed product that has been used as a feed supplement.

Skins and certain tanner-y refuse have been treated in. an alkaline medium toproduce hydrolyzed. products. This is usually accomplished by adding magnesia andbasic magnesia carbonate to produce a solution having an alkalinity not greater than a pH- of 9.5.

None of these processes are capable of hydrolyzing certain typesof hair, such ashog hair.

It has also been proposed by Ward in his Patent No. 2,474,339 to hydrolyze certain material such as chicken feathers, using ethanol, water and mercaptoethanol at. 80 C. This patent also refers to a treatment of hog hair Where the hair is treated with ethyl alcohol, Water and sodium hydroxide at a pH of 8.5, heatedfor a period of 4 hours at 130 C. in: a sealed vessel- Binkley, in his Patent No. 2,542,984 disclosesa process. for the hydrolysis of keratin materials, which. comprises analcohol, water and an acidwhich is heatedat a tern perature, preferably 1510 C. to 150 C., under pressure;v The pH of this solution is approximately 2.5- to. 4.

The present application deals with. a group of materials known as scleroproteins, which include both col- 3,177,193 Patented. Apr. 6, 1965.

"ice

by the synthetic fibers and is now a waste product on the market. Many attempts have been made to hydrolyze hog hair, so as to use it as a feed supplement, but allot these attempts haveproven failures, because of the great difliculty in hydrolyzing the particular protein contained within this material, so as to render itsuitable as a feed supplement.

It is an: object of the present invention to provide a process that will produce a food protein.

It is also an object of this invention to produce from hog hair a hydrolyzed protein that is valuable. as a feed supplement.

It is a still further object of this invention to provide a process that will hydrolyze scleroproteins, particularly keratin proteins, such as woolor hog hair to produce amino acids that are digestible and useful as a feed supplement.

It is a still further object of this invention to provide a process for hydrolyzing hairat room temperature, without pressure, in a short length of time and to maintain the maximum amount of. cyst-inc in the resulting product.

It is a still further object of this invention to avoid the use of mercapto compounds to solubilize keratin protein-s.

These and other objects will be apparent to one skilled in the art from the various examples given below, but

this invention is not limited by these example, but is only limited by the claims appended hereto.

FIGURE 1 is a graph showing the rate of solubilization of hog hair.

FIGURE 2 is a chromatographic amino acid analysis of treated hog hair.

FIGURE 3': is a graphic representation of the. efiect of reaction time on the amino acid composition of treated hog hair.

FIGURE 4 is a graphic representation of the growth rate of rats maintained on soyabean meal and modified hog hair protein.

It has been found that hog hair will hydrolze at room temperatures without pressure in 30 minutes or less when it is suspended, preferably with agitation, in an alcohol, preferably isopropyl alcohol, and sodium hydroxide if the pH is at least 11.5. i

A pH of 10 does not give satisfactory results even under increased. pressure. A pH of 11.5 or greater does give satisfactory results.

= complete solubilization; nosolubilization. b Room Temperature. Accomplished with dilute hydrochloric acid.

lagen and keratin type proteins. This group'is noted for their extreme resistance to chemical agents, and includes skin, intestines and hair. Hog hair and Wool are keratin type proteins, and particuar-ly hog hair is noted for its extreme resistance to chemical attack. All of these materials are not attacked by trypsin and are therefore of no food value.

Hog'hair has been a by-product of the packing industry for many years and has found various uses up to the present time. It hasbeen replaced, in most of these uses Prior to treatment, the hog hair was Washed and the lipid material removed. It will be noted from the tables (1 I and 3) that the pH at which solubilization occurs appears to be critical.

Various alkaline. materials have been tried, sodium hydroxide and potassium hydroxide have given satisfactory results. Ammonium hydroxide does not produce satisfactory results. a

Ammonium hydroxide (NH OH) solutions were examined, for their effect on hog hair. Various conditions were employed, as summarized in Table 2. However, under none of these conditions was any visible alterat on in the hog hair noted.

a 4 following table. 'Five percent (w./v.) hog hair was hydrolyzed with 2 N NaOI-ltalcohol (75:25) at room temperature, no agitation.

a complete solubllization; no solubilizatlon.

b Room temperature. Ammonium hydroxide within thecritical pH of 12 to 14 failed to give any satisfactory results. Reducing agents such as sodium sultite at pH of 12.6 and a temperature, of 34 C. and a digestion period of 17% hours, resulted in a 76% solubilization of hog hair. The addition of 5 mole of urea to this mixture resulted in little additional solubilization. It willbe noted from Table 3 that regardlessof the concentration of sodium hydroxide the pH of the reaction solution is the most critical factor. Apparently the destruction of the peptide linkages takes place between a pH of 11 and 12 with no solubilization occurring below a pH of 11.

Table 3 pH VERSUS CONCENTRATION Ratio Hair Concen- Diges- Temper- Hog Hair to Solvent tration tion ature, Adjusted Digested,

Time, C. Percent Hr.

1: 0. 5 24 36 I 11. 0 l: 0. 75 24 36 11. 0 0 1: 1. 0 24 36 11. 0 0 1: 1. 0 24 36 ll. 0 O 1: 0. 75 36 12. 0 22. 0 1: 1. 0 6 36 12. 0 36. 2 1: 0. 5 17 36 12. 6 92. 0 1: 0. 5 24 36 12. 5 93. 4 1: 1. 0 24 36 12. 5 96. 6

* Aqueous sodium hydroxide.

Washed, lipid-extracted hair was suspended in 0.5 M

thioglycolic acid solution in a ratio of 1:40. Urea was added to the suspension to a concentration of 10 M and, the entire solution adjusted to pH 12.6. The entire mass was allowed to react for 17 /2 hours at C. This resulted in 89.2% solubilization. The conditions of digestion'were identical to those employed for the reaction be, tween Na SO and hair, in which case only 76.4% of the hair was hydrolyzed. It appears that thioglycolic acid is a more eflicient reducing and hydrolyzing agent. Howcharacter of this compound prevent it from being con-. sidered commercially. i The above experimental conditions were repeated with niereaptoethanol replacing thioglycolic acid. Under these conditions only 24.8% of the hog hair was solubilized in the prescribed time period of 17 /2 hours.

Itfhas been found that hog hair will hydrolyze with by the presence of certain alcohols and the combination pressure.

The following alcohols shown in Table '4 have been tried I The etfect of various alcohols'on the alkaline hydrolysis of hog hairisexpressedin percent digestion in the Table 4 PERCENT OF HOG HAIR HYDROLYZED Time of Hydrolysis in Hours Alcohol. p

Table 5 SOLUBILIZATION OF HO G HAIR BY IS OPROPYL ALCOHOL- HYDROXIDE SOLUTIONS AT ROOM TEMPERA- Sodium Isopropyl Water, 5 Sample Hydroxide, Alcohol, ml. Solubilization Time Normality ml.

25 75 No efiect 3 days. 25 I 75 Do.. 25 75 .DO. 25 75 Partial, 72 hr. swelling occurred rapidly. 25 75 Practically, complete,

12 hr. 0 100 Practically, complete,

' 96 hr. 25 75 Good, 1 hr.

25 75 Complete, hr.

0 100 Complete, 4 hr. 25 75' Complete, 5 1 hr." 50. 50, Complete, %1 hr.

Three-phase solution developed. b Two-phase solution developed.

propylalcohol (3:1 ratio) for 15 to 20 minutes causesthe ever, the extreme reaction conditions, and the disagreeable 1 parts of 2 Nsodium hydroxide, is shown in FIG. 1 as 'fcurve' 1, while curve 2 is the rate of hydrolysis of hog hair -in 2 NisQdium 'hydroxide without the isopropyl alcohol. It can be readily seen from these curves that isopropyl yalco'hol'increases the'rate of hydrolysis. The availability 5 of the protein material as a feed supplement may be estiof the sodium hydroxide and alcohol as a solvent allowed the action to take place at room temperature and normal mated by digestion :ofthe hydrolyzedproduct with trypsin or pepsin; I 7

In order to determine the minimum timerequiredfor the solvent'2 N sodium hydroxide, isopropyl alcohol (3 :1

.7 ratio) to alter. the keratin fibers sufficiently to render them pepsin digestible,v hoghair Was treated .for various time periods after which the solutions wereacidified and centrifuged, the residue was suspended in an acid solution pH 2.0 and crystallized pepsin was added to. the suspen- 5 sion to give an E59 (enzyme-substrate) ratio of, 1:50.

Table 6 Solvent. Reaction Time, Wt. of Dry Residue Digested by Min. after Pepsin Hydroly- Pepsm, Percent sis, g.

* Experiment started with 5 g. of hog hair in each group.

It has been further found that an 13-8 ratio of 1:500 is as eifective as 1:50 and fibers are digested to the same extent, that is approximately 90%. It has also been found that an increase in the initial concentration of the fibers in the base alcohol solution does not hinder the penetration and elfectiveness of the pepsin. Suspension of hog hair fibers of 5%, 15% and were all digested to the same extent in the same time period.

Keratin fibers modified with the base alcohol solvent, precipitated with acid and subsequently dried at 3 7 C. could be stored without any appreciable adverse effectupon pepsin susceptibility.

in order to determine the optimum pH for the recovery of the maximum amount of starting material after modification with a base alcohol solvent, a series of samples were prepared and altered with. a base alcohol solvent and to this suspension various quantities of hydrochloric acid were added. The pH of the solution and the amount of the precipitate obtained. under the varying conditions were recorded. These results are summarized in Table 7. It is apparent from this study that an acidic environmentis necessary to. precipitate the heterogeneous mixtures of peptides. required for maximum recovery.

Table 7 Alkaline solutions have -a tendency to destroy cystine/cysteine with the formation of hydrogen sulfide. Insofar as there is very little methionine present in the keratin molecule of hog hair, it has been assumed that 5 case, the extent of destruction appears to be negligible.

A chromatographic analysis of the amino acid present in hydrolyzed hog hair is set forth in FIGURE 2.

Five percent suspension of hog hair was treated for '30 minutes with a2 N NaOH-isopropyl alcohol (75:25) at room temperature. The recation was stopped and the modified hoghair recovered by adjusting solution pH to 6=0 with concentrated HCl. The amino acid analysis was conducted on the recovered modified hog hair by hydrolyzing with HCl and resolving the resulting amino acid mixture with a descending chromatographic tech.- nique using a system consisting of What-manNo. 1' filter paper and solvent mixture consisting of sec. butanol. formic acidand' water' (75:15:10). The amino acids were located and identified on the filter paper. stripwith the use of a ninhydrin spray and standard amino acids. The density of the dye at each spot relates directly to the amount of amino acid present. The filter paper strip with the dyed resolved amino acid spots is subsequently evaluated with an Analytrol densitometer. The densitometer describes a curve from the filter paper strip whichrelates the density of the amino acid spot to the area under the curve. The curve presented is therefore a qualitative and a semiqualit-ativeamino acid analysis of the modified hog hair.

FIGURE 3 is a chromatographic determination of the composition of hydrolyzed hog hair after various lengths of times of hydrolyses showing. the efifect, particularly with reference to the cystine/cysteine content of the era posure of these amino acids to the alkaline hydrolyzing medium.

The reaction procedure and amino acid analysis procedure are identical to those described in FIGURE 2. The stability of cystine/cysteine which is of particular importance to the" nutritional value of the finished product and which is'readily'destroyed" in a basic solution, is evaluated in these curves. The curves" illustrate the importance of exactly defining the processing conditions. Treating a 5" percent hog hair suspension in a 2 N NaOH-isopropyl alcohol solvent (75 25) at roomitemperature'withont agitation causes no destruction of: cystine/ cysteine for 30minutes. After 40-? minutes the cystine/cysteine spot disappears which is indicative ofthe destruction of this". amino :acid. These curves also illustrate that there is no destruction of the base sensitive amino acids, arginine, aspartic acid", threonine and serine after minutes of treatment.

Under these conditions, the reaction time must not exceed 30 minutes.

Prior to this time it is visually evident that the individuali hair fibers are swollen and very soft and easily disintegrated. Experiments have therefore demonstrated that using the above mentioned conditions the reaction time could be reduced to l520 minutes if the hair suspension is agitated. Agitation can be accomplished with a blender or a mechanically driven stirring rod.

The tests on animals using a hog hair'feed supplement prepared as outlined above has demonstrated that the theformation of hydrogen sulfide results primarily from the destruction of cystine/ cysteine. A cadmium chlorideiodometric method for the determination. of hydrogen sulfide was employed in this study. It was established that 3 hours of contact. with the alcohol-base solvent will result-in 0;47% destruction of cystine/cysteine by this technique. This value increased to 1.75% destruction when the S-g. hog hair sample was treated in the solvent for 34 hour and. boiled for l minute in an acidic solution to remove the hydrogen sulfide formed. In either protein obtained was an acceptable feed supplement. The substitution of modified hog air protein for 20% of the protein in the. standard soyabean meal diet showed that this feed was a satisfactory feed for the growth of animals. Feed tests on growing animals demonstrates that extended digestion periods from% hour-,to 7 hours, in the modification of hog hair reaction time, in isop'nopyl alcohol 2 N sodium hydroxide did not destroy all the biological value of hog hair;

The growth rate of rats on a diet containing 20% of protein as modified hog hair is set forth in FIGURE 4. 7

Eleven ZI-day-old Sprague- Dawley rats were used for each diet. The diets were. preparedfrom'a standard protein-free diet (NutritionalBiochemical Corp.) to which soyabean meal protein (SBMP) was added. The control hair. The prepared hog hair had a composition in accord- 7 ance with the description in FIGURE 3. The final analysis of the data is presented in the accompanying table. It is to be noted that there is no statistical difference between the two groups.

Table 8 v The lower layer in the N sodium hydroxide isopropyl alcohol system contained the protein. Numerous attempts were made to recover the protein material and the sodium hydroxide solvent. The only method which suc- The feeding test was repeated with White Leghorn l-daycessfully removed the protein was acidification of the soluold cockerels. The basic diet was obtained from the Nution with hydrochloric acid. The proteins were removed tr-itional Biochemical Corporation. As in the previous 7 100%; however, the solvent was not recoverable. Meth v test the control diet contained 10 pencent SMP and the ods which were tried without success were boiling,"cold test diet 8.0 percent SMP and 2.0 percent modified hog temperature, coagulation, sodium alginate precipitation, hair. The results of the test are summarized in the followand ozone precipitation. 7 ing table. Onceagain no statistically significant difier- The loss of the sodium hydroxide solution can be parences occur between thetwogroups. tially offset by the fact than 20 and suspensions of 7 Table 9 GROWTH RATES OF CHICKS FED MODIFIED HOG HAIR DIETS Group Number Av. Final Standard Wt. Range, t Proba- Chicks Wt., g. Deviation g. Value bility 10% SBMP. 20 56.4 14. 5 70. 9-41. 9 0. 22v 0. 5 iaii r i 20 59. 1 19. 3 78. 4-39. 8

OTHER TISSUES Intestines and skin method for rendering tissues. and could replace present 7 procedures.

Specifically, the following tests were performed. Initial efiorts were devoted to determining whether these tissues can be processed "with this solvent and whether fat, protein, and solvent can be recovered in separate fractions.

The tissues were ground and the moisture and fat content were determined. The skin containing 31% solids and the intestines 60% solids. On a dry weight basis each. tissue containedapproximately 64% fat,.of' which approximately 6% was in the free fatty acid form. As in the case with hog hair, a 10% suspension of either-intestines or skin was prepared in a 2 N sodium hydroxide isopropyl alcohol (3:1) solution and digested for approximately 2 hours. The protein was precipitatedby the-addi? tion of acid. to a pH within the range'of 2:6. This protein was used in feeding testssimilar to thatobtained from the other examples. Addition of solid sodium hydroxide. to "the, 2 N sodium hydroxide isopropyl alcohol solution to form ,a 5 N solution resulted in the formation of two' liquid phases. Advantage was taken of this fact in order to separate the fat from the protein in these tissues after. 2 to 4 hours ofdigestionl The isopropyl'alcohol layer was. separated and dried. :The isopropyl alcohol was readily removed by evaporation and could be subsequently condensedand' recovered. '.Depending upon the time. required for evaporation, it was determined that the fat could be recovered as fat' or saponitied to the free fatty acid. Saponification results from .the small amount of sodium hydroxide solution,.whicl1. is miscible with the isopropyl alcohol under these conditions. from'the tissue and recovered aseither the fat or, the free fatty acids.

In eithercase, the ,fat can be entirely removed,

tissue can be successfully processed in 2 hours. Furthermore, the most recent studies indicate that a 1 N is sufiicient to'hydrolyze thezp'rotein. I

Ammonium hydroxidewas tried in place of sodium hydroxide. However, at all concentrations tried (0.5 to 15 N), no effect was observed. These results are similar to those previously obtained with hog hair.

Experiments demonstrate that in a 2 N or 1 N sodiunr hydroxide isopropyl alcohol (3:1) system, the fat floats to the surface after 1. hourof mixing. In the case of the intestine the fat forms globules the size of sodium,hy-.

droxide pellets, whereas the .skin forms a fat layer. In either. case the fat can readily be removed by skimming the surface, and the resulting product is unsaponified fat. This method isthe preferred procedure.

Hog liver and kidneytissue Experiments. have shown that homogenized hog liver and kidney tissueis readily hydrolyzed .by thisprocedure.

Specifically a 10 percent suspension, of either of these tissues are reacted'with a 2 N 'NaOH isopropyl alcohol (75 :25) solvent for a half-hour. Almost percent) complete solubilization occurs'in this time period. "Upon addit on of either HCl or H 50 *and adjustment of the 8011111011 to pH 6.0 a whitish flocculent precipitate is formed which is peptide in nature. "The protein recovered 1S1. useful as a feed supplement just as in the other exampes. v ..i.i M1,: ,7

Critical factors; p (1) Solvent: 2 N sodium hydroxide and isopropyl alcohol (75 :25); Note: Mix prior to use and allow to cool to room. temperature; Other alcohol can be used, however, isopropyl was found to be most eifective.

(2) Concentration and time of agitation: The suspension of hog hair in the'solvent can vary'from 5 to 20 percent depending uponthe extent. of agitation. Without agitation a 5 percent suspension is recommended, and

areaction time of one-half hour. A Spercent' solution with agitation can be digested in ZO minutes. Twenty percent suspension. can readily be digested a blender 'in the. prescribed time period.

3. Temperature: Roomtemperatuie'lZ l C.) is preferred. I-Iightemperature will digest the hair faster; however, considerable amino acid damage will occur; Tem-fl 9 perature in the range of 15 C. can be used successfully if the reaction time is prolonged.

(4) pH: Sodium hydroxide solution ranging from a pH of 12, that is 0.01 N to a pH of 14, that is 1 N to N, were found to function satisfactorily, although a 2 N solution is preferred.

Nature of precipitates Stopping the reaction and lowering the pH of the solution to pH 6.0 with either HCl or H 80 (concentrated or 1:1 dilution) produces a precipitate. The amount recovered is approximately 75 percent of the original material. Complete recovery can be achieved if the solution pH is further lowered to pH 2.0.

The precipitate which is obtained at pH 6.0 is characterized by a highly charged polymeric material, grey in color with a great tendency to coalesce. The precipitated mass is further characterized by a very rubbery, spongy consistency. This mass can be washed and squeezed and it still retains its spongy character.

The protein which is recovered between pH 6.0 and 2.0 is white and fiocculent.

The nature of the precipitate can be changed from a spongy form to a fiocculent form if the reaction mixture is diluted one fold with water prior to the addition of the acid.

Studies have also been performed to demonstrate that FeCl can react with the modified hair fibers prior to addition of the acid. Addition of one volume of 0.02 N FeCl to the reaction mixture prior to the addition of the acid had several important effects. Namely less acid was required to neutralize the reaction mixture to pH 6.0 and secondly it is presumed that the ferric and chloride ions interacted with the ionic groups on the protein surface. The net result of this interaction is a decrease interaction between adjacent protein molecule changing the nature of the precipitate from a spongy mass to a flocculent precipitate.

The spongy mass due to its strong intermolecular protein adjoinment produces material which can be used for protein films and fibers.

This application is a continuation-in-part of applicants prior pending application Serial No. 749,049, filed July 17, 1958; and Serial No. 176,067, filed February 27, 1962, both applications now being abandoned.

What is claimed is:

1. A process for the preparation of a proteinaceous product which can be hydrolyzed by pepsin, which consists in contacting a material containing scleroprotein with a liquid consisting of a lower aliphatic alcohol and an aqueous alkali hydroxide, said liquid having a pH of 12 to 14, at ambient temperature and normal pressure, adding a mineral acid to lower the pH to 2 to 6 and separating the protein.

2. A process for the preparation of a proteinaceous product which can be hydrolyzed by pepsin, which con sists in contacting a material containing keratin with a liquid consisting of one part of isopropyl alcohol and two parts of an aqueous sodium hydroxide, said liquid having a pH of 12 to 14, at ambient temperature and normal pressure, adding a mineral acid thereto to lower the pH to 2 to 6 and separating the protein.

3. A process for the preparation of a proteinaceous product high in cystinc/ cysteine ratio protein which can be hydrolyzed by pepsin, which consists in contacting a material containing scleroproteins with a liquid consisting I of a lower aliphatic alcohol and an aqueous alkali hydroxide, said liquid having a pH of 12 to 14, at ambient temperature and normal pressure for a length of time not exceeding 30 minutes, adding a mineral acid thereto to lower the pH to 2 to 6 and separating the protein.

4. A process for the preparation of a proteinaceous product having a high cystine/ cysteine ratio which is hydrolyzable by pepsin, which consists in contacting a material containing keratin with a liquid consisting of isoprop-yl alcohol and an aqueous sodium hydroxide solution, said liquid having a pH of 12 to 14, at ambient temperature and pressure, with agitation, for a period of time not exceeding 30 minutes, adding a mineral acid thereto to lower the pH to 2 to 6, and separating the protein.

5. A protein having a high cystine/ cysteine ratio which is characterized by being hydrolyzable by pepsin, said protein being prepared by contacting a keratin containing material with an aqueous solution consisting of an aqueous al :ali hydroxide soiution and a lower aliphatic alcohol at a pH of 12 to 14, at ambient temperature and normal pressure for a period of time not exceeding 30 minutes, suflicient to dissolve a keratin containing material, precipitating the protein by reducing the pH to 2 to 6 and separating the protein.

References Cited by the Examiner UNITED STATES PATENTS 2,474,339 7 6/49 Ward et al 260-l23.7 2,542,984 2/51 Binkley 260-423] 2,993,794 7/61 Moshy 260l23.7

FOREIGN PATENTS 556,721 10/43 Great Britain.

OTHER REFERENCES Schoberl: C.A., vol. 40, page 639 (1946).

References Cited by the Applicant UNITED STATES PATENTS 2,474,339 6/ 49 Ward et al. 2,542,984 2/ 51 Binlsley. 2,993,794 7/61 Moshy.

FOREIGN PATENTS 556,721 10/ 43 Great Britain. 7 OTHER REFERENCES Schoberl: Chemical Abstracts, vol. 40, page 639 (1946) L. I. BERCOVITZ, Primary Examiner. 

1. A PROCESS FOR THE PREPARATION OF A PROTEINACEOUS PRODUCT WHICH CAN BE HYDROLYZED BY PEPSIN, WHICH CONSISTS IN CONTACTING A MATERIAL CONTAINING SCLEROPROTEIN WITH A LIQUID CONSISTING OF A LOWER ALIPHATIC ALCOHOL AND AN AQUEOUS ALKALI HYDROXIDE, SAID LIQUID HAVING A PH OF 12 TO 14, AT AMBIENT TEMPERATURE AND NORMAL PRESSURE, ADDING A MINERAL ACID TO LOWER THE PH TO 2 TO 6 AND SEPARATING THE PROTEIN. 